Detecting Cell-Specific Gene Expression

The essence of multicellularity is the ability to express only certain portions of the genome in particular cells at particular times. This is done by the synthesis and assembly of transcription factors that turn on (and off) specific genes as required.

Many methods exist that enable one to detect a change in the whole organism as a result of gene expression in one part of it. For example, expression of the insulin gene in the beta cells of the islets of Langerhans can be monitored by measuring the level of insulin in the blood.

But there are no several ways in which the pattern of gene expression within individual cells can be monitored.

Tissue-Specific Proteins

Most genes are expressed as proteins. The synthesis within a cell of a particular protein can be detected by antibodies able to bind to that protein. If the antibodies are coupled to a fluorescent or radioactive tracer, the cells making that protein will reveal themselves.

Messenger RNA

The first step in gene expression is the transcription of DNA into RNA. A molecule of single-stranded DNA complementary to a messenger RNA (mRNA) molecule will bind to it by Watson-Crick base pairing. If the DNA is radioactive, it will identify cells making that message.

This autoradiogram (courtesy of Philip Ingham) shows regions in the Drosophila embryo that have been labeled by radioactive DNA complementary to the sequence of the mRNA for the homeobox gene fushi-tarazu (ftz). It reveals 7 bands encircling the blastoderm. These represent regions that alternate with the 7 bands formed by the even-skipped (eve) gene (lower picture).

Reporter Genes

lacZ

An example:

• the Z gene for beta-galactosidase
• even-skipped (eve), (another homeobox gene).

Any cell with the transcription factors for turning on the even-skipped promoter will begin to make beta-galactosidase. Given the proper substrate, the enzyme produces a colored product.

This photomicrograph (courtesy of Peter A. Lawrence and Blackwell Scientific Publications, Ltd.) shows 7 bands of this colored product identifying the cells that were expressing the even-skipped gene. This event was "reported" by the lacZ gene. The 7 dark stripes reveal regions that alternate with the 7 bands formed by the cells expressing fushi-tarazu (top picture).

Green fluorescent protein (GFP)

In nature, green fluorescent protein (GFP) is produced by, Aequorea victoria, the Pacific Northwest jellyfish. The protein has become of great interest to cell and molecular biologists because it can reveal gene expression in living cells.

This is done by fusing the gene for GFP to the gene whose expression you are interested in. When that gene is turned on in a cell, not only is its protein synthesized, but GFP is synthesized as well. Illuminating the cells with near-ultraviolet light causes them to fluoresce a bright green. In this way, the experimenter can see when and where the gene is expressed in the living organism.

DNA Chips

All the methods described so far are limited to monitoring the expression of one or, at most, a few genes. But as conditions change in a cell, the transcription and translation of literally hundreds of genes may be altered.

• semiconductor chip technology
• automated synthesis of oligodeoxynucleotides
• automated fluorescence scanners
• computer software,

• mammalian cells when they are transferred from a "minimal" culture medium to one enriched in growth factors
• the skeletal muscles of mice as they age.

The Chip

• Examine published gene sequences.
• For each gene, pick out ~20 different stretches of ~25 nucleotides that seem characteristic of that gene.
• Synthesize oligodeoxynucleotides corresponding to these.
• Also synthesize oligodeoxynucleotides for each of the above that have one nucleotide altered (usually near the middle). These will provide a control.
• Using robotic chip-making machines, spot these oligonucleotides individually in arrays, each spot receiving millions of copies that are fixed to the chip surface.

The Assay

• Harvest your cells. Presumably they are expressing a characteristic subset of their genes; that is, transcribing them into messenger RNA (mRNA) molecules.
• Extract the RNA.
• Make complementary DNA (cDNA) by treating the RNA mixture with reverse transcriptase.
• Transcribe the cDNA back into now much-amplified RNA.
• Attach fluorescent tags to the RNA.
• Flood the chip with this mixture.
• RNAs finding their complementary sequences on the chip will bind to them. (They will bind less strongly to adjacent spots with the single-nucleotide change if the binding is truly specific.
• Illuminate the chip and automatically record the intensities of the color at each spot.
• Use a computer to analyze the pattern.

Some results of monitoring genome-wide expression

• C-K Lee and colleagues examined the expression of 6347 mouse genes in the skeletal muscles of
  • young vs. old mice
  • well-nourished vs. mice on a calorie-restricted diet
  They report in Science (27 Aug 1999) that as mice age,
  • about 1% of their genes get turned down. These are mostly genes involved in metabolism and biosynthesis.
  • about the same number of genes get expressed more vigorously. These tend to be genes involved in the response of cells to stress.
  • Mice raised on a restricted diet did not show such dramatic shifts in gene expression as they aged. (This fits well with well-established data that mice on restricted diets age more slowly than those on rich diets.)
• When human fibroblasts (the precursors of connective tissue) are transferred from a "minimal" medium to one enriched in growth factors, they
  • not only turn on genes needed for mitosis but also
  • genes needed for wound healing and an immune response.

  This work was reported by V. R. Iyer, et al in the 1 January 1999 issue of Science. It involved the monitoring the expression of 6347 different genes.

• Monitoring gene expression in yeast shows that different combinations of transcription factors participate in turning on (and off) entire sets of genes (reported by Holstege, F. C. P., et al, in Cell, 25 November 1998.